**1. Introduction**

112 Biogas

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Yue, Z.B., Yu, H.Q. and Wang, Z.L. (2007). Anaerobic digestion of cattail with rumen culture

Yusof, N., Haraguchi, A., Hassan, M.A., Othman, M.R., Wakisaka, M. and Shirai, Y. (2009).

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As many countries have taken advantage of the richness of crude oil, fossil fuels have become the main energy source, and human activities have become entirely dependent on petroleum products. However, this is not sustainable because of the huge environmental cost of harvesting and utilizing vast amounts of fossil fuels (Fairley, 2011). Therefore, the need for alternative fuels has become critical, especially for a new generation of advanced biofuels that can maximize petroleum (crude oil) displacement and minimize the side effects of burning fossil fuels. The primary objective is then to produce biofuels from corn stalks or other 'cellulosic plants' (or even from municipal garbage) and jet fuels from dedicated energy crops such as the fast-growing *Camelina sativa* (Fairley, 2011). The challenges are then to develop the agriculture for these plants and improve their utilization at an industrial scale. In this way, net reductions in petroleum use and greenhouse-gas emissions will be long-lasting and ethical. Bridging this gap will require continued investment, research, government regulations and development of technology. The International Energy Agency (IEA) has recommend the maximized use of farm, forestry and municipal wastes as well as increased cultivation of dedicated energy crops away from lands that provide carbon sequestration and other critical environmental services. One way to develop biofuels along an environmental friendly path is to draft a set of standards and practices that biofuels producers must comply with, either voluntarily or by mandate (Fairley, 2011).

In large cities, such as Mexico City with a population of more than 20 million, concerns about waste disposal and the use of alternative energy sources has steadily increased. This population produces a tremendous amount of solid waste, more than 12,000 tons per day. On the other hand, to provide sufficient food for this population, many markets are distributed throughout the city. The central market for food distribution in Mexico City, Central de Abasto (CEDA), is the second largest market in the world, receiving 25,000 tons of food products and producing 895 tons of organic solid waste each day (84% of the total solid waste produced is organic waste, 50% of that is from fruits and vegetables).

Biogas Production and

with a subsequent biological treatment.

Cleanup by Biofiltration for a Potential Use as an Alternative Energy Source 115

liquid/gas contact (Krumdieck et al., 2008). However, the main drawbacks of these chemical technologies are the high energy requirement, the stability and selectivity of the chemicals used, the high cost of the chemicals and their regeneration, the negative environmental impacts from liquid wastes, the large equipment size requirements and the high equipment

Biological treatments are cost effective and environmentally friendly processes (Shareenfdeen et al., 2003, Ng et al., 2004, Maestre et al., 2010). Biofiltration is one of the most promising clean technologies for reducing emissions of malodorous gases and other pollutants into the atmosphere (van Groenestijn and Hesselink, 1993, van Groenestijn and Kraakman, 2005). This technology has been proven to effectively control reduced sulfur compounds in diluted gas streams (Yang et al., 1994, Smet et al., 1998, Ergas et al., 1995, Chung et al., 1996, Devinny et al., 1999; Gabriel and Deshusses, 2003, Kim and Deshusses, 2005). However, the elimination of H2S from fuel gases requires systems that can handle high loads of pollutants for extended periods of time (Maestre et al., 2010). Surprisingly, there is still a limited number of reports on the removal of high concentrations of H2S (>1000 ppmv) using biofilters, biotrickling filters and bioscrubbers. On the other hand, two processes have been effectively applied for the removal of high concentrations of H2S from biogas or fuel gas in industrial processes: the Thiopaq process (Paques, The Netherlands) and the Biopuric process (Biothane, USA). The first one is a chemical process that uses a conventional caustic scrubber and an expanded bed bioreactor for the recovery of spent caustic and elemental sulfur generation. The Biopuric process combines a chemical scrubber

Although H2S treatment for industrial processes has already been applied through the above-mentioned commercial systems, there is a need for the development of alternative and sustainable biological processes. Regarding the development of biofiltration and/or biotrickling filter systems to eliminate high H2S concentrations, Rattanapan et al., 2009, compared the elimination of 200 to 4000 ppmv of H2S in two biofiltration systems. One of the biofilters was a sulfide oxidizing bacterium immobilized on Granular Activated Carbon (GAC) (biofilter A) and the other was GAC without cell immobilization (biofilter B). The results showed that in the GAC system, the H2S was autocatalytically oxidized when it absorbed into the CAG, reaching a removal percentage of 85%. The removal was enhanced to over 98% (even at a concentration as high as 4000 ppmv) through the biological activity in biofilter A. In this last system, the maximum elimination capacity was approximately 125 gH2S/m3GAC h. In addition, Fortuny et al., 2008, reported the performance of a biotrickling filter system for treating high concentrations of H2S in simulated biogas using a single reactor. Two laboratory-scale biotrickling filters filled with different packing materials were evaluated, the inlet H2S concentration ranged from 900 to 12000 ppmv. During long-term operation, a removal percentage of 90% was determined with an extremely high H2S concentration (6000 ppmv). Maximum elimination capacities of 280 and 250 g H2S/m3 h were obtained at empty-bed residence times of 167 and 180 s, respectively. During this study, the main end products of the biological oxidation of H2S were sulfate and elemental sulfur; the final percentage of these products varied as a function of the ratio of O2/H2S supplied (v/v). At a value of 5.3, corresponding to an inlet H2S concentration of 3000, the main product was sulfate (60-70%), whereas at the higher H2S concentration of 6000 ppmv, the sulfate recovery decreased to 20-30%. Elemental sulfur production varied inversely with

corrosion rate (Tippayawong and Thanompngchart, 2010, Fortuny et al., 2008).

Fruit and vegetable waste (FVW) is produced in large quantities in markets in many large cities (Mata-Alvarez et al., 1992; Misi and Forster, 2002; Bouallagui et al., 2003; Bouallagui et al., 2005). The application of an anaerobic digestion process for simultaneous waste treatment and renewable energy production from the organic fraction of these residues could therefore be of great interest (Bouallagui et al., 2005). The high biodegradability of FVW promotes the rapid production of volatile fatty acids (VFAs), resulting in a rapid decrease in pH, which in turn could inhibit methanogenic activity (Bouallagui et al., 2003; Bouallagui et al., 2009). A strategy to avoid the acidification of the system is the addition of cosubstrates. Data obtained during the codigestion of FVW and other substrates resulted in the design of an efficient digestion process, improving methane yields through the positive synergistic effects of the mixed materials exhibiting complementary characteristics and the supply of missing nutrients from the cosubstrate (Agdag and Sponza, 2005, Habiba et al. 2009, Bouallagui et al. 2009). In a recently published study (Garcia-Peña et al., 2011), a 30- Liter anaerobic digestion reactor operated with a mixture of FVW:MR (meat residues) (75:25) had a stable CH4 production percentage of 53 ± 2 % and a sustained pH of 6.9 ± 0*.*5 (naturally regulated) in a co-digestion process. The adequate and sustained performance and stable CH4 production were a result of an appropriate buffering capacity and highly stable operation of the experimental system. However, the biogas produced during this anaerobic process needs to be cleaned before use by eliminating a relatively high content of other compounds as CO2 and H2S.

Biogas consists of approximately 60–70% (v/v) methane (CH4), 30–40% (v/v) carbon dioxide (CO2), 1–2% (v/v) nitrogen (N2), 1000–3000 ppmv H2S, 20-30 ppmv of VFAs and 10– 30 ppmv of ammonia (NH3), depending on the organic substrate used during the anaerobic process (Angelidaki et al., 2003). Hydrogen sulfide (H2S) is one of the most commonly reported reduced sulfur compounds, and represents up to 2% (v/v) . However, this H2S concentration can be higher when a rich protein feedstock is used. H2S elimination is thus required because it reduces the life span of combustion engines by corrosion, forms SO2 upon combustion and is a malodorous and toxic compound (Angelidaki et al., 2003, Pride, 2002). Malodorous gases include mainly H2S (around 3000 ppmv) and some volatile fatty acids (VFAs).

Reducing CO2 and H2S content will significantly improve the quality of biogas. There have been many technologies developed for the separation of CO2 from gas streams, including absorption by chemical solvents, physical absorption, cryogenic separation, membrane separation and CO2 fixation by biological or chemical methods (Abatzoglou and Boivin , 2009, Granite and O'Brien, 2005). These techniques are of significant industrial importance and are generally applied during natural gas sweetening and in the removal of CO2 from flue gases of power plants.

H2S is currently removed using chemical, physical or biological methods. The most commonly used method is chemical absorption by selective amines, such as diglycolamine, monoethanolamine and methyldiethanolamine, but also by absorption into aqueous solutions, physical absorption on solid adsorbents or conversion to low-solubility metal sulfides (Horikawa et al., 2004, Osorio and Torres, 2009). Water scrubbing systems are also frequently used because of their simplicity and low cost (Kapdi et al., 2005, Rasi et al., 2008). Their use allows the production of high quality CH4 enriched gas from biogas by chemical absorption where a packed bed column and a bubble column are normally used to provide

Fruit and vegetable waste (FVW) is produced in large quantities in markets in many large cities (Mata-Alvarez et al., 1992; Misi and Forster, 2002; Bouallagui et al., 2003; Bouallagui et al., 2005). The application of an anaerobic digestion process for simultaneous waste treatment and renewable energy production from the organic fraction of these residues could therefore be of great interest (Bouallagui et al., 2005). The high biodegradability of FVW promotes the rapid production of volatile fatty acids (VFAs), resulting in a rapid decrease in pH, which in turn could inhibit methanogenic activity (Bouallagui et al., 2003; Bouallagui et al., 2009). A strategy to avoid the acidification of the system is the addition of cosubstrates. Data obtained during the codigestion of FVW and other substrates resulted in the design of an efficient digestion process, improving methane yields through the positive synergistic effects of the mixed materials exhibiting complementary characteristics and the supply of missing nutrients from the cosubstrate (Agdag and Sponza, 2005, Habiba et al. 2009, Bouallagui et al. 2009). In a recently published study (Garcia-Peña et al., 2011), a 30- Liter anaerobic digestion reactor operated with a mixture of FVW:MR (meat residues) (75:25) had a stable CH4 production percentage of 53 ± 2 % and a sustained pH of 6.9 ± 0*.*5 (naturally regulated) in a co-digestion process. The adequate and sustained performance and stable CH4 production were a result of an appropriate buffering capacity and highly stable operation of the experimental system. However, the biogas produced during this anaerobic process needs to be cleaned before use by eliminating a relatively high content of

Biogas consists of approximately 60–70% (v/v) methane (CH4), 30–40% (v/v) carbon dioxide (CO2), 1–2% (v/v) nitrogen (N2), 1000–3000 ppmv H2S, 20-30 ppmv of VFAs and 10– 30 ppmv of ammonia (NH3), depending on the organic substrate used during the anaerobic process (Angelidaki et al., 2003). Hydrogen sulfide (H2S) is one of the most commonly reported reduced sulfur compounds, and represents up to 2% (v/v) . However, this H2S concentration can be higher when a rich protein feedstock is used. H2S elimination is thus required because it reduces the life span of combustion engines by corrosion, forms SO2 upon combustion and is a malodorous and toxic compound (Angelidaki et al., 2003, Pride, 2002). Malodorous gases include mainly H2S (around 3000 ppmv) and some volatile fatty

Reducing CO2 and H2S content will significantly improve the quality of biogas. There have been many technologies developed for the separation of CO2 from gas streams, including absorption by chemical solvents, physical absorption, cryogenic separation, membrane separation and CO2 fixation by biological or chemical methods (Abatzoglou and Boivin , 2009, Granite and O'Brien, 2005). These techniques are of significant industrial importance and are generally applied during natural gas sweetening and in the removal of CO2 from

H2S is currently removed using chemical, physical or biological methods. The most commonly used method is chemical absorption by selective amines, such as diglycolamine, monoethanolamine and methyldiethanolamine, but also by absorption into aqueous solutions, physical absorption on solid adsorbents or conversion to low-solubility metal sulfides (Horikawa et al., 2004, Osorio and Torres, 2009). Water scrubbing systems are also frequently used because of their simplicity and low cost (Kapdi et al., 2005, Rasi et al., 2008). Their use allows the production of high quality CH4 enriched gas from biogas by chemical absorption where a packed bed column and a bubble column are normally used to provide

other compounds as CO2 and H2S.

acids (VFAs).

flue gases of power plants.

liquid/gas contact (Krumdieck et al., 2008). However, the main drawbacks of these chemical technologies are the high energy requirement, the stability and selectivity of the chemicals used, the high cost of the chemicals and their regeneration, the negative environmental impacts from liquid wastes, the large equipment size requirements and the high equipment corrosion rate (Tippayawong and Thanompngchart, 2010, Fortuny et al., 2008).

Biological treatments are cost effective and environmentally friendly processes (Shareenfdeen et al., 2003, Ng et al., 2004, Maestre et al., 2010). Biofiltration is one of the most promising clean technologies for reducing emissions of malodorous gases and other pollutants into the atmosphere (van Groenestijn and Hesselink, 1993, van Groenestijn and Kraakman, 2005). This technology has been proven to effectively control reduced sulfur compounds in diluted gas streams (Yang et al., 1994, Smet et al., 1998, Ergas et al., 1995, Chung et al., 1996, Devinny et al., 1999; Gabriel and Deshusses, 2003, Kim and Deshusses, 2005). However, the elimination of H2S from fuel gases requires systems that can handle high loads of pollutants for extended periods of time (Maestre et al., 2010). Surprisingly, there is still a limited number of reports on the removal of high concentrations of H2S (>1000 ppmv) using biofilters, biotrickling filters and bioscrubbers. On the other hand, two processes have been effectively applied for the removal of high concentrations of H2S from biogas or fuel gas in industrial processes: the Thiopaq process (Paques, The Netherlands) and the Biopuric process (Biothane, USA). The first one is a chemical process that uses a conventional caustic scrubber and an expanded bed bioreactor for the recovery of spent caustic and elemental sulfur generation. The Biopuric process combines a chemical scrubber with a subsequent biological treatment.

Although H2S treatment for industrial processes has already been applied through the above-mentioned commercial systems, there is a need for the development of alternative and sustainable biological processes. Regarding the development of biofiltration and/or biotrickling filter systems to eliminate high H2S concentrations, Rattanapan et al., 2009, compared the elimination of 200 to 4000 ppmv of H2S in two biofiltration systems. One of the biofilters was a sulfide oxidizing bacterium immobilized on Granular Activated Carbon (GAC) (biofilter A) and the other was GAC without cell immobilization (biofilter B). The results showed that in the GAC system, the H2S was autocatalytically oxidized when it absorbed into the CAG, reaching a removal percentage of 85%. The removal was enhanced to over 98% (even at a concentration as high as 4000 ppmv) through the biological activity in biofilter A. In this last system, the maximum elimination capacity was approximately 125 gH2S/m3GAC h. In addition, Fortuny et al., 2008, reported the performance of a biotrickling filter system for treating high concentrations of H2S in simulated biogas using a single reactor. Two laboratory-scale biotrickling filters filled with different packing materials were evaluated, the inlet H2S concentration ranged from 900 to 12000 ppmv. During long-term operation, a removal percentage of 90% was determined with an extremely high H2S concentration (6000 ppmv). Maximum elimination capacities of 280 and 250 g H2S/m3 h were obtained at empty-bed residence times of 167 and 180 s, respectively. During this study, the main end products of the biological oxidation of H2S were sulfate and elemental sulfur; the final percentage of these products varied as a function of the ratio of O2/H2S supplied (v/v). At a value of 5.3, corresponding to an inlet H2S concentration of 3000, the main product was sulfate (60-70%), whereas at the higher H2S concentration of 6000 ppmv, the sulfate recovery decreased to 20-30%. Elemental sulfur production varied inversely with

Biogas Production and

Cleanup by Biofiltration for a Potential Use as an Alternative Energy Source 117

facultative anaerobes were also found. The anaerobic microorganisms were associated with higher H2S concentrations (inlet) with lower oxygen availability. The distribution of a microbial community was associated with changes in the dissolved oxygen (DO) concentration, and the accumulation of elemental sulfur and the pH (Maestre et al., 2010). Recently, Omri et al., 2011 studied the microbial community structure of the three layers (bottom, middle and top) of a biofilter using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The results obtained showed a high microbial diversity for bacteria, with the relative diversity of the bacterial community represented by the number of peaks in the profiles. Significant differences were observed between the microbial communities of the three layers of the biofilter. The Simpsons diversity index was used to determine the microbial diversity in the system, and the results indicated that the bottom and middle layers exhibited high diversity (1/D of 13.6 and 10.8, respectively). However, the microbial distribution in the top layer (1/D=8.75) was associated with the vertical gradient of the substrate, as higher H2S concentrations near the inlet allowed the growth of sulfur-oxidizing bacteria and low pH provided a favorable environment for the oxidation of H2S. The predominant bacteria in samples of the operation were found to be *Pseudomonas* sp, *Moraxellacea*, *Acinetobacter* and *Exiguobacterium* belonging

In the present chapter, the data obtained for the potential use of FVW and meat residues for methane production will be presented. The results demonstrating how a codigestion process of FVW and MR enhanced methane production by increasing the C/N ratio and controlling the natural pH in a 30L reactor will also be analyzed and discussed. At different stages of the start up of the anaerobic digestion system, methane production increased from 14 to 50% as a result of the use of a protein rich feedstock (MR). However, the H2S concentration also increased in the biogas stream under these conditions. Due to the increased H2S content, and considering that this compound does not allow for the efficient use of methane as fuel, a biofiltration system was evaluated in the elimination of H2S. The results obtained for the elimination of H2S and VFAs (average concentrations of 1500 ppmv and less than 10 ppmv, respectively) in the gas stream from an anaerobic process by a biofiltration system will then be presented. The microbial population in the biofilter when operating at steady state

Microbial culture in the biofiltration system was adapted to 10% (v/v) acetic acid and 40 mM Na2SO3.5H2O in mineral media and used for VFA and H2S degradation experiments, respectively. In situ immobilization in the biofilter was facilitated by recirculating the solution containing the microbial culture previously adapted (Ramirez-Saenz et al., 2009).

The anaerobic digestion process was evaluated in an anaerobic digestion reactor (ADR) consisting of a stainless steel tubular reactor with a total volume of 30 L into which 20 L of a (50:50) mixture of FVW and meat residues (MR) was initially packed. After the initial period

to the phyla Pseudomonadaceae, gamma-Proteobacteria and Firmicutes.

conditions is also presented and discussed.

**2. Materials and methods** 

**2.1 Microbial culture** 

**2.2 Experimental setup** 

**2.2.1 Anaerobic digestion system** 

the O2/H2S supplied (v/v), it was low at a ratio of 5.3 and increased up to 68-78% as the ratio decreased.

In a biofiltration system, a gas stream is passed through a packed bed on which pollutantdegrading organisms are immobilized as biofilms. Biotrickling filters use the same principle, but an additional liquid phase will flow through the reactor. In both systems, the microorganisms in the biofilms transform the absorbed H2S by metabolic activity into elemental sulfur or sulfate depending on the amount of available oxygen. Oxygen is thus the key parameter that controls the level of oxidation. Sulfur production (Eq. 1) results from the partial oxidation of sulfide instead of complete oxidation to sulfate (Eq. 2) when oxygen is limited, as is shown in Equations 1 and 2 (Kennes and Veiga, 2001).

$$\text{H}\_2\text{S} + 0.5\text{O}\_2 \rightarrow \text{S}\_0 + \text{H}\_2\text{O} \tag{1}$$

$$\rm H\_2S + 2O\_2 \quad \cdot \rm SO\_4^{\cdot 2} + 2H + \tag{2}$$

As the performance of a biofiltration system depends on the microbial community present in the reactor, the determination of the microorganism and the microbial activity responsible for the behavior of the process is very important. However, there is still a lack of understanding of the structure and dynamics of microbial communities and the physiological role of the main microbial population as well as the correlation between the global performance of the system with the metabolic activities of the microorganisms involved in the process. This knowledge could allow control of the reactor behavior and the design of enhanced processes to eliminate high concentrations of H2S in the gas phase because the performance of the process depends on the robustness of the microbial communities (Maestre et al., 2010).

Some authors have characterized microbial population diversity present in different gas phase reactors by analysis of biomarkers such as phospholipid fatty acids (Webster et al., 1997), molecular techniques such as fluorescent *in situ* hybridization (FISH) (Moller et al., 1996), cloning and sequencing of ribosomal RNA genes (Roy et al., 2003), terminal restriction fragment length polymorphism (Maestre et al., 2009) and denaturing gradient gel electrophoresis (Borin et al., 2006). There are only a few studies in the literature that focused on determining the microbial diversity of microorganisms capable of removing reduced sulfur compounds in biofilters or gas phase bioreactors using molecular biological approaches. Ding et al., 2006, reported the changes in the microbial diversity of a biofiltertreating methanol and H2S. In this study, the biofilter's initial microbial community had a high diversity, but after the biofiltration system was fed with H2S, the microbial diversity decreased to adapt to the low pH and use H2S as an energy source. Maestre et al., 2010 studied and described the bacterial composition of a lab-scale biotrickling filter (BTF) treating high loads of H2S using 16S rRNA gene clone libraries. The authors reported the diversity, the community structure and the changes in the microbial population on days 42 and 189 of reactor operation. The main changes in microbial diversity were observed at the beginning of the process and again when steady state operation was reached (i.e., neutral pH and at an inlet H2S concentration of 2000 ppmv). At steady state, the major sequences associated with SOB included *Thiothrix* spp., *Thiobacillus* spp., and *Sulfurimonas denitrificans*. Additionally, FISH analysis was used to determine the spatial distribution of sulfuroxidizing bacteria (SOB) along the length of the reactor under pseudo-steady state operation. The aerobic species were found to be predominantly along the system, but some facultative anaerobes were also found. The anaerobic microorganisms were associated with higher H2S concentrations (inlet) with lower oxygen availability. The distribution of a microbial community was associated with changes in the dissolved oxygen (DO) concentration, and the accumulation of elemental sulfur and the pH (Maestre et al., 2010). Recently, Omri et al., 2011 studied the microbial community structure of the three layers (bottom, middle and top) of a biofilter using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The results obtained showed a high microbial diversity for bacteria, with the relative diversity of the bacterial community represented by the number of peaks in the profiles. Significant differences were observed between the microbial communities of the three layers of the biofilter. The Simpsons diversity index was used to determine the microbial diversity in the system, and the results indicated that the bottom and middle layers exhibited high diversity (1/D of 13.6 and 10.8, respectively). However, the microbial distribution in the top layer (1/D=8.75) was associated with the vertical gradient of the substrate, as higher H2S concentrations near the inlet allowed the growth of sulfur-oxidizing bacteria and low pH provided a favorable environment for the oxidation of H2S. The predominant bacteria in samples of the operation were found to be *Pseudomonas* sp, *Moraxellacea*, *Acinetobacter* and *Exiguobacterium* belonging to the phyla Pseudomonadaceae, gamma-Proteobacteria and Firmicutes.

In the present chapter, the data obtained for the potential use of FVW and meat residues for methane production will be presented. The results demonstrating how a codigestion process of FVW and MR enhanced methane production by increasing the C/N ratio and controlling the natural pH in a 30L reactor will also be analyzed and discussed. At different stages of the start up of the anaerobic digestion system, methane production increased from 14 to 50% as a result of the use of a protein rich feedstock (MR). However, the H2S concentration also increased in the biogas stream under these conditions. Due to the increased H2S content, and considering that this compound does not allow for the efficient use of methane as fuel, a biofiltration system was evaluated in the elimination of H2S. The results obtained for the elimination of H2S and VFAs (average concentrations of 1500 ppmv and less than 10 ppmv, respectively) in the gas stream from an anaerobic process by a biofiltration system will then be presented. The microbial population in the biofilter when operating at steady state conditions is also presented and discussed.
